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at the BRCA1-NBR2 locus
Emilie Auriol, Lise-Marie Billard, Frédérique Magdinier, Robert Dante
To cite this version:
Emilie Auriol, Lise-Marie Billard, Frédérique Magdinier, Robert Dante. Specific binding of the methyl
binding domain protein 2 at the BRCA1-NBR2 locus. Nucleic Acids Research, Oxford University
Press, 2005, 33 (13), pp.4243-4254. �10.1093/nar/gki729�. �hal-01663899�
Specific binding of the methyl binding domain
protein 2 at the BRCA1-NBR2 locus
Emilie Auriol, Lise-Marie Billard, Fre´de´rique Magdinier
1and Robert Dante*
Laboratoire de Ge´ne´tique et Cancer, FRE2692 CNRS, UCBL1, 8 avenue Rockefeller, 69373 Lyon cedex 08,
France and1Laboratory of Molecular Embryology, NIH/NICHD, Bethesda, MD, USA
Received April 15, 2005; Revised and Accepted July 5, 2005
ABSTRACT
The methyl-CpG binding domain (MBD) proteins are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitu-tively methylated region of a CpG island containing the bidirectional promoter of the Breast cancer pre-disposition gene 1, BRCA1, and the Near BRCA1 2 (NBR2 ) gene. In HeLa cells, quantitative chromatin immunoprecipitation assays indicated that MBD2 is associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. MBD2 depletion (90%), mediated by a transgene express-ing a small interferexpress-ing RNA (siRNA), did not induce MeCP2 or MBD1 binding at the methylated area. Furthermore, the lack of MBD2 at the BRCA1-NBR2 CpG island is associated with an elevated level of NBR2 transcripts and with a significant reduction of induced-DNA-hypomethylation response. In MBD2 knockdown cells, transient expression of a Mbd2 cDNA, refractory to siRNA-mediated decay, shifted down the NBR2 mRNA level to that observed in unmodified HeLa cells. Variations in MBD2 levels did not affect BRCA1 expression despite its stimula-tion by DNA hypomethylastimula-tion. Collectively, our data indicate that MBD2 has specific targets and its presence at these targets is indispensable for gene repression.
INTRODUCTION
In mammals, DNA methylation at CpG islands located within regulatory regions is a crucial event in gene silencing. The various mechanisms leading to methylation-dependent down-regulation of the transcription remain to be fully determined.
However, the discovery of methyl-CpG binding domain (MBD) proteins and their interacting partners provides a direct link between DNA methylation and the establishment of a repressive chromatin architecture (1). The five MBD proteins identified to date share the functional MBD (2). Four of them, MBD1, MBD2, MBD3 and MeCP2, are directly involved in the transcriptional repression of methylated templates in vertebrates and, with the exception of MBD3, bind methylated DNA (3).
MeCP2, the founding member of the MBD family, represses transcription through its interactions with the histone deacetylase–Sin3 complex and the histone 3 lysine 9 trimethyl-transferase Suv39H1 (4–6). MBD3 is part of the histone deacetylase and chromatin remodeling Mi2/NuRD complex, which is targeted to methylated templates in the MeCP1 complex by MBD2 (7,8). In mouse, an interaction between the MBD2 and the Sin3 complex has also been described previously (9). The HDAC complex associated with MBD1 is not yet identified (10), but other MBD1 partners such as histone 3 lysine 9 dimethyl transferase SETDB1 and the chromatin assembly protein 1 have been characterized previously (11,12).
In cell lines, MBD1, MBD2 and MeCP2 transiently repress the expression of genes driven by either strong or weak promoters (13), suggesting some functional redundancies between these proteins. Moreover, both the MBD1 and the
MeCP2 proteins are associated,in vivo, with the differentially
methylated region of the mouse imprintedU2af1-rs1 gene (14)
and, in cancer cell lines, some methylated CpG islands are bound by multiple MBD proteins (15). Since many cell and tissue types express multiple MBD proteins, these data suggest that other members of the family might compensate the absence of a specific MBD. However, functional specificities
have been observed. In human, MeCP2 mutations are the
cause of the RETT syndrome (RTT), a neurological disorder associated with motor function impairment that represents one of the most common causes of mental retardation in females
(16). In mouse, loss of Mecp2 leads to phenotypes that
*To whom correspondence should be addressed. Tel:+33 4 78 78 59 22; Fax: +33 4 78 78 27 20; Email: dante@univ-lyon1.fr Present addresses:
Emilie Auriol and Robert Dante, Unite´ INSERM 590, Laboratoire d’Oncologie Mole´culaire, Centre Le´on Be´rard, 28 rue Laennec, 69373 Lyon Cedex 08, France Fre´de´rique Magdinier, Laboratoire de Biologie Mole´culaire de la Cellule, CNRS UMR 5161-ENS, Lyon, France
The Author 2005. Published by Oxford University Press. All rights reserved.
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Nucleic Acids Research, 2005, Vol. 33, No. 13 4243–4254
resemble some of the symptoms of RTT patients (17,18), indicating a specific function of MeCP2 in the maturation
of central nervous system. Mbd1/ mice also show
neuronal maturation deficits associated with hippocampal functions (19). Therefore, the involvement of these proteins in different pathways of the central nervous system argues against a functional redundancy between Mecp2 and Mbd1. In addition, mice lacking Mbd2 do not exhibit specific pheno-type except a maternal behavior deficit (20).
Although no global misexpression of endogenous methy-lated genes has been detected in the first studies, candidate gene approaches have identified some specific target genes. In mouse and rat, Mecp2 is directly involved in a depolarization-controlled repression of the brain derived neurotrophic factor gene in neurons (21,22), and that of
the imprinted DLX5 gene driven at a distance by Mecp2
has also been shown previously (23). Specific repression is
not restricted to MeCP2. In mouse, loss ofMbd1 induces small
but significant increase in the expression of the endogenous virus, IAP, associated with chromosome instability in cultured
neuron cells (19).Mbd2/ mice exhibit a disordered helper
T cells differentiation owing to ectopic expression of the IL4 gene (24).
A comparative study in human cancer cell lines using a chromatin immunoprecipitation (ChIP) assay combined with a CpG island microarray also suggests some specificities, since some cancer-associated hypermethylated genes are bound by multiple MBD proteins while others are associated with a single MBD protein (15). Furthermore, in interphase MCF7 cells, the distribution of MeCP2 does not parallel that of methylated cytosine and heterochromatin, and a select-ive binding of MeCP2 to some repetitselect-ive elements has also been observed previously (25). The mechanisms driving the MBD binding are not yet fully determined. The relative abundance of the MBD proteins might be an important para-meter, since it was suggested that there are more methyl-CpGs in the genome than MBD protein molecules (1), and whether MBD1, MBD2 and MeCP2 are randomly associated with sites or segregate owing to other constraints is not yet known.
To address this question, we investigated in HeLa cells, the MBD proteins binding pattern at a CpG island located in the
50 end of the Breast cancer predisposition gene 1 (BRCA1).
BRCA1 lies head to head with the Near BRCA1 2 gene (NBR2) (26). Experiments of site-directed deletions led to the identi-fication of a bidirectional promoter that is embedded within a large CpG island (13). This CpG island contains a region that is constitutively methylated in all human cell lines and tissues, except gametes and preimplantation embryos (27–29). Therefore, this model represents an interesting tool for inves-tigating MBD proteins binding specificity and their effects on transcription at a naturally methylated CpG island.
MATERIALS AND METHODS Cell culture
Cervix cancer cell line (HeLa) was obtained from ATCC (Rockville, MD) and grown in minimum essential medium (Eagle; Sigma, L’isle d’Abeau, France) supplemented with
10% fetal calf serum. Cells were grown at 37C in a humidified
5% CO2atmosphere.
5-Aza-20-deoxycytidine treatments
For 5-aza-20-deoxycytidine (5-aza-dC; Sigma) treatments, cell
lines were seeded at low density (3–4 · 105 cells/100 mm
dish) 16 h before treatment with a final concentration of 10mM
5-aza-dC. The medium was changed after 24 h drug addition and every subsequent day. RNAs were isolated after 72 h. Cell transfections
pCMV-MeCP2-HA (kindly provided by Dr A. Bird), pRev-MBD2 (modified from pCMV-MBD2, kindly provided by Dr A. Bird) and pGL3 basic (Promega, Lyon, France) were transfected using the Exgen500 transfection reagent, according to the manufacturer’s instructions (Euromedex, Mundolsheim, France). Cells were collected 48 h after trans-fection. Transfection efficiency was tested using a CMV-lacZ
vector andb-galactosidase activity was observed in >90% of
the cells.
Quantification of nucleic acids
Nucleic acids were quantified by densitometry using the Fluorimeter Fluors and the Quantity One software (BioRad,
Ivry, France), either from agarose gel containing 1 mg/ml
ethidium bromide or dots mixed with an equal volume of a 200-fold diluted solution of RiboGreen (Molecular Probes, Interchim, Montlu¸con, France).
RNA extraction and quantitative RT–PCR
Total RNA was extracted from cell lines with the RNeasy kit (Qiagen, Courtaboeuf, France) and treated with DNase I using the DNA-free kit (Ambion Inc., Cambridgeshire, UK). After extraction, the integrity of total RNA was examined on a 1.2%
agarose gel containing 1mg/ml ethidium bromide and
quan-tified. cDNA fragment ofNBR2 (from position+146 to +604
from the transcription start site) was amplified by RT–PCR
using forward 50-GAGGTCTCCAGTTTCGGTAA-30 and
reverse 50-GAACCAAGGTGAAGGACCAA-30. After
clon-ing the PCR product into a pGEM-T easy vector (Promega),
a 109 bp long deletion was performed within theNBR2 cDNA
using the restriction endonuclease MaeIII (Roche, Meylan, France). The competitor RNA was synthesized using the Sp6 RNA polymerase according to the manufacturer’s instruc-tions (Promega). After DNase I digestion (Ambion Inc.) and RNeasy purification (Qiagen), this competitor RNA was quan-tified by densitometry and then diluted in the presence of yeast tRNA as a carrier (40 ng/ml; Ambion). For BRCA1 and MBD2 quantitative RT–PCR, previously designed competitors were used (30,31).
Equal amounts of total RNA samples (0.6 mg for NBR2,
0.3 mg for BRCA1 and 0.1 mg for MBD2) were coamplified
with increasing amounts of competitor RNAs in a final volume
of 50ml using the One Step RT–PCR kit, Q solution for NBR2
(Qiagen) and 0.6mM of each primers. After a 30 min
incuba-tion at 50C, RT was inactivated by heating at 95C for
15 min. The PCR amplification was then accomplished in a thermocycler under the following conditions: 35 cycles, 30 s
denaturation at 94C, 1 min annealing at 62C forNBR2 and
MBD2, 64C forBRCA1 and 1 min 30 s extension at 72C. In
addition, control experiments for each competitor RNA were performed by omitting the RT to ensure that the signal was the result of RNA and not DNA amplification. PCR products were
analyzed on a 2% agarose gel and quantified. Then, the normalized signals corresponding to the target mRNA and the competitor were plotted against the initial number of competitor molecules added to the test tubes. The abscissa of the intersection of the curves represents an estimation of the equivalence point between the initial amount of the com-petitor molecules and the number of copies of the mRNA assayed (32).
Protein extraction and western blot analysis
Forty micrograms of whole cell lysates were separated by electrophoresis through SDS–10% polyacrylamide gels and analyzed by western blot (31). For the detection of the MBD proteins, rabbit polyclonal antibodies against MBD2 (kindly provided by Dr P. Wade) or MeCP2 (Upstate Biotech-nology, Lake Placid, NY) and mouse monoclonal antibody against MBD1 (Abgent, San Diego, CA), were diluted 1/2000. The secondary anti-rabbit-HRP or anti-mouse-HRP conjugate antibodies were diluted 1/5000. The immunocom-plexes were detected using the ECL system (Amersham, Saclay, France).
Quantitative chromatin immunoprecipitation
Cells were washed and scraped off the culture dishes in phosphate-buffered saline, and nuclei were prepared in ice-cold hypotonic buffer (10 mM Tris–HCl, pH 7.4, 10 mM NaCl and 5 mM MgCl2). Each step was performed on ice and in
the presence of a mixture of protease inhibitors (Complete;
Roche). After centrifugation, cells were lyzed in the hypotonic buffer containing 0.1% NP-40. Nuclei were harvested by
cen-trifugation at 2000 r.p.m. (700 g) and were washed in the
same buffer. Nuclear proteins were then cross-linked to DNA by an incubation with 1% formaldehyde for 10 min
at room temperature and then for 40 min at +4C.
Cross-linking was stopped by adding 125 mM glycine for 5 min. After centrifugation, the pellets were washed in the hypotonic buffer and resuspended in 1–2 ml of SDS lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris–HCl, pH 8). Nucleo-protein complexes were sonicated to reduce the length of DNA fragments to 300–600 bp. Insoluble material was removed and the supernatant was collected. Thirty microliters of this frac-tion was preserved as an input control and the rest was diluted 1:10 in ChIP dilution buffer (ChIP assay Kit; Upstate Biotech-nology). The chromatin solution was precleared for 1 h by
incubation with 80 ml of salmon sperm DNA–protein A–
agarose beads (Upstate Biotechnology). The soluble fraction
was collected and 15ml of monoclonal anti-MBD1 (Abgent),
polyclonal anti-MBD1 (Abcam, Cambridge, UK), anti-MBD2 (kindly provided by Dr P. Wade), anti-MeCP2 (Upstate) and anti-mouse IgG (Dakocytomation, Trappes, France) antibod-ies were added and incubated overnight. Then, for ChIP
monoclonal antibodies, 1 mg of rabbit polyclonal
anti-mouse antibody (Dakocytomation) was added and incubated for 1 h. After immunoprecipitation, immune complexes were
collected by adding 60 ml of salmon sperm DNA–protein
A–agarose beads for 1 h. The supernatant corresponding to the unbound fraction was collected. After washing (accord-ing to the manufacturer’s instructions), complexes were
eluted from the beads in 1% SDS and 0.1 M NaHCO3. This
fraction corresponds to the bound (anti-MBD) or the
non-MBD-specific antibodies (anti-mouse IgG) fractions.
Cross-links were reversed by heating samples at 65C in
200 mM NaCl. DNA was recovered by proteinase K digestion, phenol extraction and ethanol precipitation. Finally, DNA samples from the input, unbound, non-MBD-specific antibody and bound fractions were quantified.
Quantitative PCR amplification was performed using a
competitor DNA. DNA fragment of theBRCA1-NBR2 CpG
island (from position 1244 to 1004 from the BRCA1
transcription start site, fragment A) was amplified by PCR
using forward 50-GCTTTTCGCCCACTCGGTCC-30 and
reverse 50-CAGAGCTGGCAGCGGACGGT-30. After
clon-ing the PCR product into a pGEM-T easy vector (Promega), a single cut within the island fragment was performed using AccIII and an insertion of a 43 bp chemically synthesized duplex-oligonucleotide was accomplished using T4 DNA ligase (Roche). After cloning and purification (Plasmid Maxi Kit; Qiagen), this competitor vector was quantified. Equal amounts (2 ng) of total DNA samples from the input, unbound and bound fractions were coamplified with
increas-ing amounts of competitor plasmid in a final volume of 100ml
using the HotStarTaq polymerase kit (Qiagen) and 0.4mM of
each primer. The PCR amplification was accomplished after
activation of theTaq polymerase (15 min at 95C and 35 cycles
in a thermocycler under the following conditions: 30 s
dena-turation at 94C, 1 min annealing at 63C and 1 min 30 s
extension at 72C). PCR products were analyzed on a 2%
agarose gel containing 1mg/ml ethidium bromide and were
quantified by densitometry as described. Then, the normalized
signals corresponding to the BRCA1 CpG island fragment
and the competitor were plotted against the initial quantity of competitor molecules added to the test tubes. The abscissa of the intersection of the curves represents an estimation of the equivalence point between the initial amount of the competitor
and the quantity of theBRCA1-NBR2 CpG island fragment in
each sample (Supplementary Figure 8s).
We also analyzed three other regions of the methylated BRCA1-NBR2 CpG island (B, C and D fragments) by perform-ing a semi-quantitative PCR. We amplified equal amounts of total DNA samples (0.5 ng) from the input, unbound
and bound fractions, using forward 50
-AAGGGCTCCTC-CAGCACGGC-30 and reverse 50
-TTCTGAGGGACCGA-GTGGGC-30 for the B fragment (from positions 1364 to
1218 from the BRCA1 transcription start site); forward
50-TTCAAGCGGGGTGCAGGCGG-30 and reverse 50
-CCC-TCTCTGGGCTGGCCGAA-30 for the C fragment (from
positions 1389 to 1637); forward 50
-CTGGTGCATA-TAAAATCCTCAGGC-30 and reverse 50
-GCACAGGG-CAAGGCTCAGGA-30 for the D fragment (from positions
1653 to 1928).
As a control, we amplified an unmethylated region
includ-ing the promoter and the exon 1 of theBRCA1 gene (‘promoter
fragment’ from position 56 bp to +263 bp) using forward
50-TAGCCCTTGGGTTCCGTG-30and reverse 50
-TCACAA-CGCCTTACGCCTC-30.
PCR was accomplished in a final volume of 100ml using the
HotStar Taq polymerase kit (Qiagen) and 0.4 mM of each
primer, as described previously, under the following
condi-tions: 30 s denaturation at 94C, 1 min annealing at 64C for
the fragment B, 66C for the fragments C and D or at 60C
PCR products were analyzed and quantified by densitometry.
Then, the signals corresponding to the BRCA1-NBR2 CpG
island fragment in the input, unbound and bound fractions were compared.
Stable knockdown of MBD2 by short interfering RNA and transient reversion
HeLa cells stably expressing small interfering RNA (siRNA) were established as described previously (33). Briefly, the
pSUPERMBD2 plasmid was generated by cloning the 19 nt
sequence 50-GGAGGAAGTGATCCGAAAA-30 (beginning
575 nt from the translation start site in the mouse MBD2
mRNA), separated by a spacer from its reverse complement as a BglII/HindIII fragment (synthesized at Proligo, Paris, France) into the pSUPER vector. This vector directs synthesis of RNA from the polymerase III-H1-RNA promoter that is processed in the cell to siRNA. One microgram of
pSU-PERMBD2was cotransfected with 0.1mg of a plasmid encoding
a geneticin resistance gene into HeLa cells by using Lipofec-tamine 2000 (Invitrogen, Cergy Pontoise, France). Cells were
selected using 1mg/ml G418 (Roche) for 15 days. Clones were
picked and expanded for an additional 20 days and were
ana-lyzed for MBD2 mRNA and protein levels as described
previously.
Transient reversion of RNA interference (RNAi) was performed by transfecting HeLa clones with a pRev-MBD2
vector in which Mbd2 cDNA sequence was modified at the
siRNA-target site. Despite five point mutations (50-GGAAGA
GGTCATTCGCAAA-30, mutated nucleotides are underlined)
and also according to the genetic code, the new cDNA sequence encodes a functional Mbd2 protein. This vector was generated by digesting the pCMV-MBD2 vector (kindly
provided by Dr A. Bird) within theMbd2 cDNA sequence with
KspI (Roche) and AccIII (Promega) endonucleases. This KspI/ AccIII fragment was gel-purified (MinElute gel purification Kit; Qiagen) and digested by ApaI and AccI (Roche). A 117 bp
double-stranded oligonucleotide (Eurogentec, Seraing,
Belgium), containing the five silent point mutations, was intro-duced into ApaI/AccI site to replace the initial 117 bp fragment. After cloning and sequencing (Biofidal, Vaulx-en-Velin, France), plasmids exhibiting the expected sequence were
selected. The transcription of the mutated form of Mbd2
cDNA allows this mRNA to bypass siRNA-mediated
decay. HeLa andMBD2 knockdown clones were transfected
with the resulting vector. Finally, MBD2 protein expression was assessed after cellular proteins extraction and western blotting.
Sodium bisulfite modification
The sodium bisulfite modification method, followed by the endonuclease restriction of PCR products, was used to determine the CpG methylation pattern. Sodium bisulfite converts unmethylated cytosines to uracils whereas the methylated cytosines remain unmodified. In the resultant modified DNA, uracils are replicated as thymines during PCR amplification (34).
DNA was extracted from cell lines with the QiaAmp DNA Mini Kit (Qiagen). After extraction, the integrity of total DNA was examined on a 1.2% agarose gel and quantified by densitometry. The sodium bisulfite reaction was carried
out as described previously (28). DNA was amplified using strand-specific primers designed to amplify a 304 bp region
(methylated region, 1246 to 942 bp from the BRCA1
transcription start site) and a 250 bp region (unmethylated
region, 592 to 343 bp from the BRCA1 transcription
start site) in the CpG island of the BRCA1 gene. The first
round of PCR amplification was accomplished in 100 ml
using the HotStar Taq DNA polymerase Kit (Qiagen) and
0.4 mM of the primers (methylated region: forward 50
-TTT-TGTTTTGTGTAGGGCGGTT-30 and reverse 50
-CCTTAA-CGTCCATTCTAACCGT-30; unmethylated region: forward
50-GTTTATAATTGTTGATAAGTATAAG-30 and reverse
50-CCCACTCTTTCCGCCCTAAT-30) after 15 min at 95C
forTaq polymerase activation and 35 cycles (30 s denaturation
at 94C, 1 min annealing at 55C for the methylated region
or at 56C for the unmethylated region, and 1 min 30 s
exten-sion at 72C). An aliquot of the first amplification of the
methylated region was reamplified with internal primers
(forward 50-TGAGAATTTAAGTGGGGTGT-30 and reverse
50AACCCTTCAACCCACCACTAC-30) with the same
con-ditions. PCR products were then analyzed by digestion with the restriction enzymes, CfoI (Roche) and HphI (NEN Biolabs, Saint Quentin Yvelines, France). Digestion products were
analyzed on a 2% agarose gel containing 1 mg/ml ethidium
bromide.
RESULTS
MBD2 binds to the methylated region of a CpG island flanking the bidirectional BRCA1-NBR2 promoter
A large CpG island, 2.8 kb in length, lying from nt1810 to
+974 from the BRCA1 transcription start site (Figure 1) exhibits several features, suggesting that it might be a target for MBD proteins. Indeed, this CpG island is unmethylated
in gametes, but regionally methylated (position 2000 to
1000) in all tissues and cell lines so far analyzed (27–29), and nuclease protection assays indicate that this methylated region is embedded in a condensed chromatin structure in breast cell lines (29) and in HeLa cells (data not shown).
Quantitative ChIP assays were performed using antibodies directed against MBD1, MBD2 and MeCP2. The analyzed
region (nt1244 to 1004; Figure 1, ‘A’ segment) is located
close to, but does not overlap with, the repetitive element
LTR12c (nt3125 to 1276), which is part of the CpG island
(Figure 1). Data obtained from at least three independent ChIP assays for each antibody are shown in Figure 2A. The fractions immunoprecipitated with a non-MBD protein specific anti-body (anti-mouse IgG) did not contain enough DNA for PCR assay, indicating an efficient preclearing step.
When antibodies against MBD2 are used, the amount of BRCA1-NBR2 CpG island per ng of total DNA in immuno-precipitated fraction (Figure 2A, ‘bound’) is higher (5-fold) than in input or non-retained fractions (Figure 2A, ‘input’ and ‘unbound’). Therefore, these data indicate that the BRCA1-NBR2 CpG island is specifically immunoprecipitated by the anti-MBD2 antibodies. In contrast, when antibodies against MeCP2 or MBD1 are used, the immunoprecipitated
DNA segment is less abundant in the bound fraction than in the input and the unbound fractions (Figure 2A).
A dose-dependent amplification of the ‘B’ region (nt1364
to1218) was performed on each fraction obtained from the
ChIP procedure (Figure 2B). As observed at the segment lying
from position1244 to 1004, enrichment in BRCA1-NBR2
CpG island DNA in the bound fraction is only observed when the chromatin is immunoprecipitated with anti-MBD2 antibodies, while ChIPs with anti-MeCP2 or anti-MBD1 antibodies lead to a depletion of this DNA in the bound
Figure 1. The BRCA1-NBR2 locus. The BRCA1 gene is located head to head with the NBR2 gene and the two genes are separated by a bidirectionnal promoter (25). Transcription start sites of both genes are arrowed in black. Black box,BRCA1 and NBR2 exons. The locus includes a CpG island of 2784 bp in length (%G+C, 57; ObsCpG/ExpCpG, 0.65; CpGProD software, http://pbil.univ-lyon1.fr). M+, constitutively methylated region of the CpG island (28); light gray box, LTR12c retroelement; dark gray box, AluY sequence (Repeat Masker software, version 2002). Black lines represent the positions of the fragments amplified by A, competitive PCR; B, C, D and ‘promoter’, semi-quantitative PCR, after ChIP. Positions are indicated from theBRCA1 transcription start site.
Figure 2. MBD2 associates the methylated region of the BRCA1 CpG island in HeLa cells. (A) Amounts of immunoprecipitated BRCA1 island (fragment A, see Figure 1) measured by competitive PCR (error bars, SD from, at least, three independent experiments). Cross-linked chromatin was immunoprecipitated using rabbit polyclonal anti-MBD2, anti-MeCP2, anti-MBD1 or monoclonal mouse anti-MBD1 antibodies. Purified DNA from the input, unbound or bound fractions was quantified and quantitative PCR analysis was accomplished. Then, the intensity of the bands corresponding to the PCR products was plotted against the initial number of competitor molecules as described in Supplementary Data, Figure 8s. Black box, amount ofBRCA1 island fragments per ng of the input DNA. Open box, amount ofBRCA1 island fragments per ng of the unbound DNA. Gray box, amount of BRCA1 island fragments per ng of the bound DNA. (B) Three other regions of theBRCA1 CpG island were analyzed by ChIP. These regions, fragments B, C and D (Figure 1), are located upstream the first region amplified by competitive PCR. After immunoprecipitation, purified DNA from the different fractions was quantified and 0.5 ng of this DNA was amplified. The intensities of the bands corresponding to the PCR products amplified from the input, unbound and bound fractions are shown. (C) Representative experiment of PCR amplification of an unmethylated region including the promoter and the exon 1 of theBRCA1 gene (‘promoter fragment’ from position56 to +263 bp from the BRCA1 transcription start site) on MBD2-ChIP fractions.
fractions (Figure 2B). MBD2 binding to the methylated part of the repeated LTR12c element was further investigated by
performing ChIP assays at the ‘C’ region (nt1389 to 1637)
and ‘D’ region (nt1653 to 1928). A significant enrichment
of these fragments in the chromatin fraction immunoprecipi-tated by antibodies against MBD2 was observed (Figure 2B). We also amplified an unmethylated region containing the
promoter and the exon 1 of theBRCA1 gene (from position
56 to +263 bp from the BRCA1 transcription start site).
As expected, enrichment in BRCA1-NBR2 CpG island was
not observed in the chromatin fraction immunoprecipitated with anti-MBD2 antibodies (Figure 2C), since the amplified segment is unmethylated in HeLa cells (28). Taken together, these data strongly suggest that the methylated region of
the BRCA1-NBR2 CpG island is only bound by MBD2 in
HeLa cells and that MBD2 binds only to the methylated region.
Depletion of MBD2 is not compensated by the binding of MeCP2 or MBD1 at the methylated region of BRCA1-NBR2 CpG island
HeLa cells contain 20 times more MBD2 than MeCP2
transcripts (30). In addition, it has been shown that some methylated DNA regions are bound by multiple MBD pro-teins (15). Taken together, these data suggest that the specific
binding of MBD2 to theBRCA1-NBR2 CpG island might be a
consequence of the relative concentrations of the MBD pro-teins in HeLa cells. To address this issue, MBD propro-teins bind-ing patterns at this CpG island were investigated in HeLa cells depleted in MBD2. Stable clones, expressing a siRNA targeted
to MBD2 transcripts (MBD2a and b isoforms), were
structed by stable transfection of pSUPER vector (33)
con-taining aMBD2 specific insert.
In several HeLa clones, quantitative competitive RT–PCR
assays indicated a constitutive reduction of 89–96% inMBD2
mRNA level (Figure 3A, MBD2 KD clones). Western blot
analysis also showed a dramatic decrease in MBD2 proteins
in theseMBD2 KD clones carrying the pSuper-MBD2
trans-gene (Figure 3B). In addition, persistent suppression ofMBD2
expression is conserved after many passages since efficient MBD2 knockdown is still observed after 2 months of
continu-ous culture. Furthermore, the expression ofMeCP2 and MBD1
was not affected in MBD2 knockdown clones (KD clones)
when compared with the wild-type HeLa cells (data not shown).
Quantitative ChIP assays from HeLa cells depleted in
MBD2 proteins showed noBRCA1-NBR2 CpG island
enrich-ment in the bound fraction when chromatin is immunoprecipi-tated with anti-MBD2 antibodies (Figure 4A). Furthermore, as previously observed in HeLa cells, the immunoprecipitated fractions are depleted in the methylated island when anti-MeCP2 or anti-MBD1 were used (Figure 4A). The same results were obtained when a different antibody, anti-MBD1, was used (data not shown). In addition, a positive immunoprecipi-tation of MeCP2-bound chromatin has been obtained in another set of experiments (Figure 7A and B), and in KD cells additional ChIP control experiments indicated that MBD1 proteins were associated with juxtacentromeric
sequences Sat2 (data not shown). Thus, in MBD2-depleted
HeLa cells, the methylated region (nt 1244 to 1004) of
BRCA1-NBR2 CpG island is not bound by these other two MBD proteins. This observation was extended to the adjacent
methylated sequences (nt 1364 to 1218) using a
semi-quantitative approach (Figure 4B). Taken together, these
data indicate that the methylated region of theBRCA1 island
remains free from MeCP2 or MBD1 in MBD2-depleted HeLa cells, suggesting that MBD2 binding is specific.
Transcriptional repression of the BRCA1-NBR2 locus by MBD2
The bidirectional promoter, driving both BRCA1 and NBR2
expression, is relatively far (1 kb) from the methylated domain of the CpG island. However, in human cell lines,
chemically induced demethylation stimulates BRCA1 gene
expression andin vitro methylated expression vectors carrying
this CpG island is repressed when transiently transfected
into human cell lines (28). Since NBR2 and BRCA1 share
the same promoter region (Figure 1), we tested a potential
role of DNA methylation in the expression of the NBR2
gene. In HeLa cells, demethylation was induced by
5-aza-dC treatments. NBR2 transcripts were then quantified
using a competitive RT–PCR method (a representative assay is shown in Figure 5A). As described previously (28), 5-aza-dC
treatment of HeLa cells elevated BRCA1 expression by
1.8-fold and an overexpression of NBR2 (3.1-fold) was also
observed (Figure 5B).
These data raised the question whether MBD2 is a
methylation-dependent repressor of the BRCA1-NBR2
Figure 3. MBD2 mRNA and protein quantification in HeLa cells expressing stable siRNA (MBD2 knockdown cells). (A) Quantification of MBD2 mRNA by competitive RT–PCR assay in wild-type HeLa and three clones ofMBD2 knockdown cells (a, b and c) performed from 0.1mg of total RNA mixed with increasing amount of competitorMBD2 RNA. The intensity of the bands corresponding to the PCR products was plotted against the initial number of competitor molecules in order to estimate the amount ofMBD2 mRNA present in 1mg of each sample. (B) Immunoblot analysis of MBD2 proteins in HeLa and knockdown cell lines. MBD2 proteins were probed using a rabbit polyclonal antibody. Then, the same membrane was probed using a mouseb-tubulin antibody as a loading control.
Figure 4. MeCP2 and MBD1 do not compensate for MBD2 depletion at the BRCA1-NBR2 CpG island in MBD2 knockdown HeLa cells. (A) Amounts of immunoprecipitatedBRCA1 island (fragment A, Figure 1) measured by competitive PCR (error bars, SD from, at least, three independent experiments). Black box, amount ofBRCA1 island fragments per ng of the input DNA. Open box, amount of BRCA1 island fragments per ng of the unbound DNA. Gray box, amount of BRCA1 island fragments per ng of the bound DNA. (B) Representative examples of BRCA1 semi-quantitative PCR (fragment B, Figure 1) on MBDs-ChIP assays (as described in Figure 2B).
Figure 5. Effects of 5-aza-dC on the expression of BRCA1 and NBR2 in HeLa and MBD2 knockdown cells. (A) Quantitative competitive RT–PCR assay of NBR2 mRNAs was performed from 0.6mg of total RNA mixed with increasing amount of NBR2 competitor RNA. Amounts of competitor molecules: lane 1, 5 · 104
; lane 2, 105; lane 3, 5· 105; lane 4, 106; lane 5, 5· 106. The RT–PCR products were analyzed on a 2% agarose gel containing ethidium bromide. The intensity of the bands
corresponding to the PCR products was plotted against the initial number of competitor molecules. The diagrams of the intensity values are represented below the gels. Black box, 349 bp band competitorNBR2 RNA; open box, 458 bp band endogenous NBR2 RNA. (B) NBR2 and BRCA1 transcripts in HeLa cells and MBD2 KD cells treated or untreated with 10mM 5-aza-dC for 72 h. Quantitative RT–PCR analyses were performed from 0.6 mg of total RNA mixed with increasing amount of competitorNBR2 RNA or from 0.3mg of total RNA mixed with increasing amount of competitor BRCA1 RNA (32). Average values obtained from four independent experiments (error bar, SD) are shown. Control: untreated cells.
bidirectional promoter. In MBD2 depleted cells, a stimulation
ofNBR2 expression by 1.7-fold is observed when compared
with wild-type HeLa cells (Figure 5B). In addition, inMBD2
KD clones, 5-aza-dC treatments (Figure 5B) had a lower
effect (1.6-fold) onNBR2 expression than in wild-type cells
(3.1-fold). Since both cell types exhibit the same amount of NBR2 transcripts after 5-aza-dC treatments (Figure 5B), these
data suggest that MBD2 is an important factor in theNBR2
methylation-dependent control.
However, the same amounts of BRCA1 transcripts were
found in MBD2 KD clones and in wild-type HeLa cells
(Figure 5B). In addition,BRCA1 response to 5-aza-dC
treat-ment is not modified by MBD2 depletion (Figure 5B),
indi-cating that BRCA1 expression is independent of the MBD2
binding. Furthermore, control experiments indicated that
the methylation pattern ofBRCA1-NBR2 CpG island was not
modified in MBD2 KD cells when compared with the
wild-type HeLa cells (Supplementary Data, Figure 9s). Taken together, these data suggest a specific methylation-dependent
repression of NBR2 by MBD2.
Mbd2 expression rescues the reduction of NBR2 transcript in MBD2 knockdown clones
Functional control of the specific repression of NBR2 by
MBD2 has been performed by expressing the MBD2
target-cDNA in a form that is refractory to siRNA-mediated decay. Five silent point mutations of the third-codon within the
targeted region were introduced into a mouse Mbd2 cDNA
and the resulting expression vector (pRev-MBD2) was
tran-siently expressed in MBD2 KD clones after transfection.
Western blot analysis of HeLa andMBD2 KD clones indicated
that Mbd2 proteins were efficiently expressed from the pRev-MBD2 expression vector (Figure 6A).
In wild-type HeLa cells, the level of BRCA1 and NBR2
transcripts was not affected by the Mbd2 overexpression due to the pRev-MBD2 vector (Figure 6B). Also, the pCMV-MBD2 expression vector containing a non-mutated
mouseMbd2 cDNA did not alter BRCA1 and NBR2
transcrip-tion in HeLa cells (data not shown). Therefore, the amount of MBD2 is not a limiting factor in the transcriptional
repres-sion of theBRCA1-NBR2 locus, in HeLa cells. In contrast, in
MBD2 KD cells, pRev-MBD2 expression induced a 28–45%
decrease in NBR2 expression (Figure 6B) and transfected
MBD2 KD cells exhibited an NBR2 mRNA level equivalent to the level observed in wild-type HeLa cells. No statistically
significant decrease inBRCA1 mRNA level was observed in
these transfected MBD2 KD cells (Figure 6B). These data
confirm that, at the BRCA1-NBR2 locus, MBD2 specifically
represses the NBR2 gene.
Specificity of transcriptional repression by MBD2 Taken together, these data indicate that, in HeLa cells, under
physiological concentrations, MBD2 binds to the
BRCA1-NBR2 CpG island and the absence of this protein is not com-pensated by the binding of other MBD proteins. However,
experiments have suggested that exogenous BRCA1 might
be repressed by the overexpression of Mecp2 (28). We have, therefore, investigated the ability of the exogenous Mecp2 to repress the transcription of the endogenous locus. Mecp2 was overexpressed in HeLa and MBD2 KD cells by
transient transfection of the pCMV-MeCP2-Ha vector. Immunoblots indicated a high expression of Mecp2 after trans-fection in both cell types (Supplementary Data, Figure 10s).
Quantitative ChIP assays, from transfectedMBD2 KD cells,
showed a slight (1.7-fold) but statistically significant
enrich-ment in the A segenrich-ment ofBRCA1-NBR2 CpG island (Figure 1,
‘A’ segment) in the chromatin fraction immunoprecipitated by antibodies against MeCP2 (Figure 7A). Mecp2 binding was further confirmed by a semi-quantitative ChIP in the adjacent region (Figure 7B). This Mecp2 binding is associated with a
decrease (17–26%) in NBR2 transcripts (Figure 7C), when
compared toMBD2 KD cells transfected with an empty vector.
In addition, in HeLa cells, overexpression of Mecp2 did not
modify NBR2 transcription, as observed after pRev-MBD2
transfections (Figure 7C). BRCA1 mRNA amounts remain
unchanged in both wild-type and MBD2 KD HeLa cells
expressing MeCP2 at a high level (Figure 7C). Thus, in MBD2 depleted cells, elevated level of exogenous Mecp2
leads to Mecp2 binding at the BRCA1-NBR2 locus and to a
partial repression ofNBR2.
DISCUSSION
Transient transfection ofin vitro methylated expression
vec-tors has clearly demonstrated that members of the methyl-CpG binding protein family, MBD1, MBD2 and MeCP2, are directly involved in the methylation-dependent repression of
Figure 6. MBD2 transfection experiments in HeLa and MBD2 knockdown cells. (A) Immunoblot analysis of MBD2 proteins in HeLa and MBD2 KD cells transfected with an MBD2 vector expressing a transcript resistant to RNAi (pRev-MBD2 vector). HeLa andMBD2 KD cells were transfected with the pRev-MBD2 vector or with an empty pGL3 basic vector (pGL3). MBD2 proteins, from whole cell extract prepared 48 h after transfection, were probed using a rabbit polyclonal antibody. (B) Quantitative RT–PCR analysis of BRCA1 and NBR2 transcripts was performed (as described in Figure 5) from HeLa andMBD2 KD cells transfected with the pRev-MBD2 or an empty vector (pGL3). The ratio between the amounts of the transcripts in pRev-MBD2-transfected cells and pGL3-transfected cells was then calculated. Mean values obtained from at least four independent transfection experiments are shown [*P¼ 0.0049 (<0.05), t-test].
gene expression (1). In the mouse, loss of these proteins leads to a prominent effect on cerebral function (17–19), suggesting a specific dependence of these tissues on the control medi-ated by MBD proteins. Alternatively, functional redundancy between MBD proteins may be more important in other tis-sues. In line with this hypothesis, global gene overexpression was not observed in mice lacking a particular MBD protein (17–20). Nevertheless, further studies indicated that gene-specific dysregulation in non-cerebral tissues are associated with the loss of MBD proteins (21,22,24). Analysis of the chromatin proteins also raises the question of the redundancy between these proteins.
Our analysis, in the HeLa cell line, of a CpG island at the BRCA1-NBR2 locus indicates that the methylated part of this
CpG island is bound by MBD2 while MeCP2 and MBD1 are not detected in this region. Furthermore, western blot analysis, using antibodies directed against MeCP2 and MBD1 produced
a signal at the expected size (15),85 and 70 kDa,
respec-tively, indicating that both proteins are expressed in this cell line (data not shown). Thus, the lack of detection of these two
proteins at the methylated region of theBRCA1-NBR2 CpG
island is not due to their absence. In addition, the depletion
in BRCA1-NBR2 CpG island in the chromatin fractions
immunoprecipitated by the anti-MeCP2 and MBD1 antibodies also indicates that these two proteins are linked to other chro-matin domains in HeLa cells. Therefore, the methylated part of this CpG island seems to be specifically associated with MBD2 in our cellular model. Although all the MBD proteins are ubiquitously expressed in somatic tissues, variations in their expression levels, depending on the cell type and the differentiation state, have been observed previously (35). For example, in mouse brain, Mecp2 and Mbd1 are highly
expressed when compared with Mbd2 (2), whileMBD2
tran-scripts are 20-fold more abundant than MeCP2 mRNA in
breast tissues (30), suggesting that the relative MBD protein concentrations might be involved in the phenotypes associated with their experimental depletions. Therefore, their relative concentrations might be associated with a prominent binding of MBD2 to some methylated DNA segments. However, in these cells, the strong targeted-depletion of MBD2 proteins, induced by RNAi, does not induce the binding of other MBD
proteins at the methylated region of theBRCA1-NBR2 CpG
island. Thus, these data suggest that this sequence is a pref-erential target for MBD2.
Among the mechanisms potentially involved in the prefer-ential binding of MBD2, differences in affinity for methylated DNA might be an important component, since it was shown that murine Mbd2b exhibits a higher affinity than Mecp2 for methylated oligonucleotides (36). Although this possibility
cannot be excluded, it should be noted that, inMBD2
knock-down HeLa clones, MBD2 proteins were nearly undetectable and the amount of the transcripts was reduced by 92% on the average, while MeCP2 and MBD1 protein levels remained unchanged (data not shown). In addition, hypermethylated CpG islands are associated with MeCP2 in several cell lines and tissues expressing all MBD proteins (14,15,37,38) and when overexpressed in HeLa cells depleted in MBD2
proteins, Mecp2 binds to the BRCA1-NBR2 CpG island.
Taken together, these data suggest that corepressors of tran-scription leading to a non-permissive chromatin environment
may be, in vivo, of importance for the specific recruitment
of MBD2, or alternatively for the exclusion of MeCP2 and
MBD1 at theBRCA1-NBR2 locus. The possible presence of a
recently described MBD2 partner, the MIZF protein (39), has also been investigated, since the methylated region of the BRCA1-NBR2 CpG island contains a consensus binding site
for this protein at position 960 bp. However, we failed to
detect MIZF at this locus by ChIP (data not shown). In human cell lines, while chemically induced
hypomethy-lation was associated with the activation of bothBRCA1 and
NBR2, transcriptional repression mediated by MBD2 seemed
to be gene specific, since MBD2 knockdown elevated only
NBR2 transcription, in HeLa cells. In addition, this phenotype was rescued by exogenous Mbd2, indicating that this effect is a
specific consequence of theMBD2 knockdown. Furthermore,
Figure 7. MeCP2 transfection experiments in HeLa and MBD2 knockdown cells. (A) Quantitative MeCP2-ChIP assays in MBD2 KD cells transfected with the pCMV-MeCP2-HA vector. After immunoprecipitation of cross-linked chromatin with an anti- MeCP2 antibody, competitive PCR analysis (fragment A, Figure 1) was performed (as described in Figure 2). Average values obtained from two independent MeCP2-ChIP assays are shown. (B) A representative example of a BRCA1 semi-quantitative PCR (fragment B, Figure 1) performed on these ChIP assays. See Supplementary Results for immunoblot controls (Supplementary Figure 10s). (C) BRCA1 and NBR2 mRNA quantification in HeLa andMBD2 KD cells transfected with an MeCP2 vector. Four independent transfection experiments of HeLa andMBD2 KD cells with the pCMV-MeCP2-HA vector or an empty pGL3 basic vector (pGL3), RNA extraction and quantitative RT–PCR analysis were performed. Average values are presented on this graph [*P¼ 0.018 (< 0.05), t-test].
this latter experiment also confirmed that MBD2 is not
involved inBRCA1 expression, since the expression of
exoge-nousMbd2 did not modify the amounts of BRCA1 transcripts
in both wild-type HeLa cells andMBD2 KD cells. Moreover,
the response ofNBR2 to DNA hypomethylation was reduced
by a factor of2 in MBD2-depleted HeLa cells, while BRCA1
response was not affected by the MBD2-siRNA. Although
MBD2 depletion does not completely abolish the effect of 5-aza-dC, the consistent reduction of 5-aza-dC-response in MBD2-depleted HeLa cells confirms that MBD2 significantly
contributes to the methylation-dependentNBR2 repression.
In the human genome, BRCA1 lies head to head with the
NBR2 gene (40). The transcription start site of BRCA1 is
separated from theNBR2 gene by a 218 bp bidirectional
pro-moter (40,41). This region is embedded within a large CpG island (13) and the methylated part of this region corresponds to a nuclease resistant chromatin structure in human breast cell lines (29) and in HeLa cells. Hence, this region is located at the 30end of theNBR2 transcription start site and at the 50end of BRCA1 (Figure 1). The specificity of MBD2 towards NBR2
transcription from the bidirectional BRCA1-NBR2 promoter,
suggests that the methylation-dependent repression ofNBR2
andBRCA1 is mediated by different mechanisms.
The analysis of the 50 region of the endothelin receptor B
gene in human cell lines shows that extensive methylation closely downstream of the initiation site does not abolish gene expression (42). However, studies using plasmid and episomal vectors (43–45) and patch methylation techniques indicate that methylation downstream of a promoter can decrease transcription levels, suggesting that this epigenetic mark may also affect the elongation rate. Therefore, the
reduced expression of NBR2 mediated by MBD2 might be
dependent on such mechanisms. In line with this hypothesis, BRCA1 expression is not affected by MBD2 binding to the methylated region of the CpG island upstream its promoter. Nevertheless, inhibition of DNA methylation stimulates BRCA1 expression, suggesting that its repression might be associated with the binding of different MBD proteins to some other methylated DNA segments. Alternatively, DNA methylation may have a direct effect on the binding of
regu-latory factors since it was suggested that the1582 to 202
region of the 50 end of BRCA1 contains several weak
regu-latory sites (both enhancers and repressors) with additive effects (40) and a putative cAMP-responsive element binding transcription factor binding site has been mapped in the BRCA1 promoter (46).
MBD2 belongs to a family of methylation-dependent tran-scriptional repressor and its presence in aberrantly methylated genes has been observed in human cancer cell lines (47–49). However, a direct relationship between MBD2 binding and gene repression has been established only for a few genes. In mouse, it had been shown that Gata-3 and Mbd2 act in
competition for the regulation of Il4 gene transcription in
T-helper cells (49). Evidences for a contribution of MBD2 in silencing the aberrantly methylated pi-class glutathione S-transferase (GSTP1) promoter have been obtained, in the breast cancer cell line MCF7 cells, by MBD2-depletion mediated by siRNA (48).
Functional domains have been mapped within the MBD proteins and beside their methyl-CpG binding domain, all these proteins posses a domain involved in gene repression,
the transcriptional repression domains (TRDs); in contrast to the MBD, no sequence similarities were found between the different TRDs (1). Many promoters, including Simian virus 40, cytomegalovirus and beta-actin promoters (3), are efficiently repressed by tethering the minimal TRD of MeCP2,
MBD1 and MBD2. However, it was shown that the L1
promoter is not repressed by fusion protein containing the TRD of MBD2, but is silenced by fusion proteins containing
either MeCP2 or MBD1 TRDs (50). The repression ofNBR2
by MBD2 does not seem to be specific of its TRD, since in MBD2-depleted HeLa cells, exogenous Mecp2 decreased NBR2 transcription. Similarly, inhibitory effect of exogenous
Mecp2 has already been observed in Mbd2/ mice. Indeed,
previous studies have shown that the methylation-dependent repression of reporter genes is impaired in fibroblast cell lines from these mice, suggesting that endogenous Mecp2 proteins do not compensate for the absence of Mbd2 (18). Never-theless, exogenous Mecp2 restores full repression in the
Mbd2/ cells, indicating that the overexpression of Mecp2
can counteract the absence of Mbd2 in this system (18).
AlthoughMbd2/mice are viable and fertile (20), a
reduc-tion in Mbd2 inhibits the development of intestinal adenomas
in the tumor-proneApcMin/+mouse (51). In human cell lines,
MBD2 antisense inhibitors suppress tumorigenesis in vitro and in vivo, when these cells are implanted in nude mice as a model (52). Taken together these data suggest that MBD2 represents a new target potential in cancer therapy and, therefore, new insights on MBD2 specificities are, in this context, of impor-tance. Collectively, our data indicate that MBD2 specifically
binds to theBRCA1 island and represses NBR2. In addition,
our results suggest that the relative abundance of MBD is not a major factor in their targeting to some methylated DNAs. Furthermore, the cellular model developed in this study may represent a valuable tool for the identification of MBD2 target genes, which could be valuable for therapeutics applications.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We are most grateful to Dr Paul Wade for providing MBD2 antibodies and to Dr Adrian Bird for the pCMV-MeCP2-HA and pCMV-MBD2 vectors. This work was supported by the Ligue Nationale contre le Cancer (Comite´ du Rhoˆne and Comite´ de la Loire), France and the Association pour la Recherche sur le Cancer, France. Funding to pay the Open Access publication charges for this article was provided by Centre Leon Berard, Lyon, France.
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